Primers and method of detecting bacteria

ABSTRACT

The first primer of the invention is a primer which, when used in PCR under appropriate conditions, serves to detectably amplify 16S rRNA-encoding DNAs of bacteria of the  Escherichia, Salmonella  and  Vibrio  genera, but when used in PCR under the same conditions, does not serve to detectably amplify either chloroplast 16S rRNA-encoding DNAs or mitochondrial 16S rRNA-encoding DNAs. The second primer of the invention is a primer which, when used in PCR under appropriate conditions, serves to detectably amplify 16S rRNA-encoding DNAs of  Staphylococcus aureus  and  Bacillus cereus,  but when used in PCR under the same conditions, does not serve to detectably amplify either chloroplast 16S rRNA-encoding DNAs or mitochondrial 16S rRNA-encoding DNAs.

TECHNICAL FIELD

The present invention relates to PCR primers for detecting bacteriabelonging to Escherichia, Salmonella, and Vibrio genera. The inventionalso relates to PCR primers for detecting bacteria belonging toStaphylococcus and Bacillus genera, in particular PCR primers fordetecting Staphylococcus aureus and Bacillus cereus. Further, theinvention relates to a method for detecting bacteria in foodstuffs usingthese primers.

BACKGROUND ART

In the prior art, to check the presence or absence of specific bacteria,such as foodborne pathogenic bacteria in foodstuff, bacteria areisolated from foodstuffs and cultured, and visual observation of thecolonies (for color reactions and morphology of the colonies),microscopic observation, Gram-staining, biochemical testing and othertests are made. These methods require at least 2 days to demonstrate thepresence or absence of the specific bacteria. Such methods can hardly beemployed on manufacture s voluntary inspection of foodstuffs prior toshipment thereof since even though it can successfully detect bacteria,the resulting delay in taking due measures is intolerable.

In recent years, methods have been developed which comprise isolatingbacteria from foodstuffs and culturing the same, and testing thethus-obtained colonies by the PCR method (Science, 230, 1350 (1985))using primers corresponding to DNA of specific bacterial species torapidly detect the specific bacterial DNA (Japanese Unexamined PatentPublications 1999-332600, 1995-236500, and 1993-317098). However, evenwhen these methods are used, these methods require at least one day forisolation of bacteria from foodstuffs and cultivation of them.

DNAs coding for rRNAs (ribosomal ribonucleic acids) are often used asthe targets for detection by the PCR method. This is because rRNAs existin all organisms except for viruses and show high levels of homologyamong organisms of the same species because of relatively slow rates ofevolution thereof. As regards bacterial 16S rRNA, in particular,numerous sequence data has been accumulated, therefore, DNAs encoding16S rRNA seems to suit for detecting bacteria by the PCR method.

It has also been proposed that a direct PCR assay with broad-range PCRprimers capable of hybridizing with many bacterial 16S rRNA-encodingDNAs are used for detecting a large variety of bacteria by use ofbacterial DNA extracted from test samples (J. Dent. Res. 78(4): 850-856(1999); J. Clinical Microbiology June 2000, 2076-2080; J. ClinicalMicrobiology February 1999, 464-466).

However, in cases where foodstuffs are used as test materials, the DNAsof the foodstuffs themselves are also extracted by the procedure forextracting bacterial DNAs. Therefore, if the primers are low inspecificity, those chloroplast or mitochondrial DNAs derived from thefoodstuffs, too, will be amplified, making the presence or absence ofbacteria ambiguous.

To overcome such disadvantages, it is also possible to use primerscapable of specifically hybridizing with the DNAs coding for the 16SrRNA of specific bacterial species (J. Applied Microbiology 1997, 83,727-736). In this case, however, it becomes necessary to carry out testsseparately for individual target bacterial species, hence the number oftests per foodstuff sample would increase. Thus, this method couldhardly be employed by foodstuff manufacturers in voluntary testing.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide PCR primers withwhich the presence or absence of a plurality or group of specificbacterial species, containing foodborne pathogenic bacteria causative,in foodstuffs can be detected easily and rapidly, and a method by whichthe presence or absence of specific bacteria and a specific group ofbacteria in foodstuffs can be easily and rapidly detected using thoseprimers.

The present inventors made investigations in an attempt to accomplishthe above object and made the following findings i) to iv).

-   i) When a DNA extraction procedure is carried out with a foodstuff    and, with the DNA mixture obtained, the PCR method is carried out    using primers capable of hybridizing with DNAs coding for 16S rRNAs    of plurality of bacterial species (such a DNA being hereinafter    referred to as 16S r-DNA), the possibility of the plant chloroplast    16S r-DNAs being simultaneously detected is high since bacterial 16S    r-DNAs and plant chloroplast 16S r-DNAs are similar to each other in    sequence.

Therefore, for avoiding the simultaneous detection of foodstuff-derived16S r-DNAs in the PCR method applied to DNAs extracted from foodstuffs,it is necessary to use primers serving to amplify the 16S r-DNAs of thetarget bacterial species but not serving to amplify chloroplast 16Sr-DNAs.

-   ii) Those main problem-bacteria possibly existing in foodstuffs and    causing food poisoning include a group of bacteria which consist of    bacteria of Escherichia (including O-157 and other pathogenic    Escherichia coli stains) and Salmonella genera belonging to the    Enterobacteriaceae family; bacteria of the Vibrio genus belonging to    the Vibrioaceae family; bacteria of the Staphylococcus (including    Staphylococcus aureus) and Bacillus (including Bacillus cereus)    genera. Therefore, the presence or absence of those main problem    bacteria in foodstuffs can be checked in a simple and easy manner by    carrying out the PCR procedure using primers serving to amplify DNAs    of bacteria of Escherichia, Salmonella and Vibrio genera and the PCR    procedure using primers serving to amplify DNAs of bacteria of the    Staphylococcus (in particular Staphylococcus aureus) and Bacillus    (in particular Bacillus cereus) genera.-   iii) Further, when the primers serving to amplify the 16S r-DNAs of    bacteria of the Escherichia, Salmonella and Vibrio genera do not    serve to amplify the 16S r-DNAs of bacteria other than the group of    bacteria consisting of bacteria of the Enterobacteriaceae and    Vibrioaceae families, the possibility of false-positive reactions    can be reduced. As a result, the sensitivity of detection of the    targeted bacteria can be improved. Similarly, when the primers    serving to amplify the 16S r-DNAs of bacteria of the Staphylococcus    (in particular Staphylococcus aureus) and Bacillus (in particular    Bacillus cereus) genera do not serve to amplify the 16S r-DNAs of    bacteria other than the group of bacteria consisting of bacteria of    the Staphylococcaceae and Bacillaceae families, the possibility of    false-positive reactions can be reduced. As a result, the    sensitivity of detection of the targeted bacteria can be improved.-   iv) Contaminants contained in foodstuffs may inhibit the PCR    procedure in certain instances. Therefore, when the step mentioned    below, for instance, is carried out after grinding the foodstuff    sample, the bacteria contained in the foodstuff sample can be    efficiently separated from the foodstuff sample.

When the ground foodstuff sample is centrifuged at a relatively lowspeed, namely by a centrifugal force of about 50 to 300×g, the insolubleingredient and the cell debris in the foodstuff sample mostlyprecipitate while bacteria remain in the supernatant; thus bacteria canbe separated from the foodstuff sample. The bacteria can be harvested bycentrifuging the supernatant by a centrifugal force of 2,000×g orgreater.

Therefore, when DNA is extracted from the bacteria-containing sedimentobtained by such a centrifugation procedure and the PCR is carried outusing the DNA obtained as template, simultaneous detection of DNAoriginating from the food can be avoided more effectively and, further,the influence of PCR-inhibiting contaminants possibly contained infoodstuffs can be avoided.

Investigations based on such findings as described above have now led tocompletion of the present invention, which provides the followingprimers, primer sets, PCR kit, and method for detecting bacteria.

-   1. A primer which, when used in PCR under appropriate conditions,    serves to detectably amplify 16S rRNA-encoding DNAs of bacteria of    the Escherichia, Salmonella and Vibrio genera, but when used in PCR    under the same conditions, does not serve to detectably amplify    either chloroplast 16S rRNA-encoding DNAs or mitochondrial 16S    rRNA-encoding DNAs.-   2. A primer according to Item 1 which, when used in PCR under the    above conditions, may serve to detect the group of bacteria of the    Enterobacteriaceae and Vibrioaceae families.-   3. A primer according to Item 1 or 2 which, when used in PCR under    appropriate conditions, serves to detectably amplify 16S    rRNA-encoding DNAs of bacteria of the Escherichia, Salmonella and    Vibrio genera, but when used in PCR under the same conditions, does    not serve to detectably amplify 16S rRNA-encoding DNAs of bacteria    other than the group of bacteria of the Enterobacteriaceae and    Vibrioaceae families.-   4. A primer which, when used in PCR under appropriate conditions,    serves to detectably amplify 16S rRNA-encoding DNAs of    Staphylococcus aureus and Bacillus cereus, but when used in PCR    under the same conditions, does not serve to detectably amplify    either chloroplast 16S rRNA-encoding DNAs or mitochondrial 16S    rRNA-encoding DNAs.-   5. A primer according to Item 4 which, when used in PCR under the    above conditions, may serve to detect the group of bacteria    consisting of bacteria of the Staphylococcaceae, Bacillaceae and    Aerococcaceae families.-   6. A primer according to Item 4 or 5 which, when used in PCR under    appropriate conditions, serves to detectably amplify 16S    rRNA-encoding DNAs of Staphylococcus aureus and Bacillus cereus, but    when used in PCR under the same conditions, does not serve to    detectably amplify 16S rRNA-encoding DNAs of bacteria other than the    group of bacteria consisting of bacteria of the Staphylococcaceae,    Bacillaceae and Aerococcaceae families.-   7. A primer which, when used in PCR under appropriate conditions,    serves to detectably amplify 16S rRNA-encoding DNAs of bacteria of    the Staphylococcus and Bacillus genera, but when used in PCR under    the same conditions, does not serve to detectably amplify either    chloroplast 16S rRNA-encoding DNAs or mitochondrial 16S    rRNA-encoding DNAs.-   8. A primer according to Item 7 which, when used in PCR under the    above conditions, may serve to detect the group of bacteria of    bacteria of the Staphylococcaceae, Bacillaceae and Aerococcaceae    families.-   9. A primer according to Item 7 or 8 which, when used in PCR under    the above conditions, serves to detectably amplify 16S rRNA-encoding    DNAs of bacteria of the Staphylococcus and Bacillus genera, but when    used in PCR under the same conditions, does not serve to detectably    amplify 16S rRNA-encoding DNAs of bacteria other than the group of    bacteria consisting of bacteria of the Staphylococcaceae,    Bacillaceae and Aerococcaceae families.-   10. The following primer (1) or (2):    -   (1) A primer consisting of the base sequence of base numbers 21        to 30, the base sequence of base numbers 20 to 30, the base        sequence of base numbers 19 to 30, the base sequence of base        numbers 18 to 30, the base sequence of base numbers 17 to 30,        the base sequence of base numbers 16 to 30, the base sequence of        base numbers 15 to 30, the base sequence of base numbers 14 to        30, the base sequence of base numbers 13 to 30, the base        sequence of base numbers 12 to 30, the base sequence of base        numbers 11 to 30, the base sequence of base numbers 10 to 30,        the base sequence of base numbers 9 to 30, the base sequence of        base numbers 8 to 30, the base sequence of base numbers 7 to 30,        the base sequence of base numbers 6 to 30, the base sequence of        base numbers 5 to 30, the base sequence of base numbers 4 to 30,        the base sequence of base numbers 3 to 30, the base sequence of        base numbers 2 to 30 or the base sequence of base numbers 1 to        30 in SEQ ID NO:1;    -   (2) A primer which is a DNA comprising at most 40 bases and        containing, on the 3 terminal side thereof, the base sequence of        base numbers 21 to 30, the base sequence of base numbers 20 to        30, the base sequence of base numbers 19 to 30, the base        sequence of base numbers 18 to 30, the base sequence of base        numbers 17 to 30, the base sequence of base numbers 16 to 30,        the base sequence of base numbers 15 to 30, the base sequence of        base numbers 14 to 30, the base sequence of base numbers 13 to        30, the base sequence of base numbers 12 to 30, the base        sequence of base numbers 11 to 30, the base sequence of base        numbers 10 to 30, the base sequence of base numbers 9 to 30, the        base sequence of base numbers 8 to 30, the base sequence of base        numbers 7 to 30, the base sequence of base numbers 6 to 30, the        base sequence of base numbers 5 to 30, the base sequence of base        numbers 4 to 30, the base sequence of base numbers 3 to 30, the        base sequence of base numbers 2 to 30,or the base sequence of        base numbers 1 to 30 in SEQ ID NO:1.-   11. The following primer (3) or (4):    -   (3) A primer consisting of the base sequence of base numbers 14        to 23, the base sequence of base numbers 13 to 23, the base        sequence of base numbers 12 to 23, the base sequence of base        numbers 11 to 23, the base sequence of base numbers 10 to 23,        the base sequence of base numbers 9 to 23, the base sequence of        base numbers 8 to 23, the base sequence of base numbers 7 to 23,        the base sequence of base numbers 6 to 23, the base sequence of        base numbers 5 to 23, the base sequence of base numbers 4 to 23,        the base sequence of base numbers 3 to 23, the base sequence of        base numbers 2 to 23 or the base sequence of base numbers 1 to        23 in SEQ ID NO:2;-   (4) A primer which is a DNA comprising at most 40 bases and    containing, on the 3 terminal side thereof, the base sequence of    base numbers 14 to 23, the base sequence of base numbers 13 to 23,    the base sequence of base numbers 12 to 23, the base sequence of    base numbers 11 to 23, the base sequence of base numbers 10 to 23,    the base sequence of base numbers 9 to 23, the base sequence of base    numbers 8 to 23, the base sequence of base numbers 7 to 23, the base    sequence of base numbers 6 to 23, the base sequence of base numbers    5 to 23, the base sequence of base numbers 4 to 23, the base    sequence of base numbers 3 to 23, the base sequence of base numbers    2 to 23 or the base sequence of base numbers 1 to 23 in SEQ ID NO:2.-   12. The following primer (5) or (6):    -   (5) A primer consisting of the base sequence of base numbers 15        to 24, the base sequence of base numbers 14 to 24, the base        sequence of base numbers 13 to 24, the base sequence of base        numbers 12 to 24, the base sequence of base numbers 11 to 24,        the base sequence of base numbers 10 to 24, the base sequence of        base numbers 9 to 24, the base sequence of base numbers 8 to 24,        the base sequence of base numbers 7 to 24, the base sequence of        base numbers 6 to 24, the base sequence of base numbers 5 to 24,        the base sequence of base numbers 4 to 24, the base sequence of        base numbers 3 to 24, the base sequence of base numbers 2 to 24        or the base sequence of base numbers 1 to 24 in SEQ ID NO:3;    -   (6) A primer which is a DNA comprising at most 40 bases and        containing, on the 3 terminal side thereof, the base sequence of        base numbers 15 to 24, the base sequence of base numbers 14 to        24, the base sequence of base numbers 13 to 24, the base        sequence of base numbers 12 to 24, the base sequence of base        numbers 11 to 24, the base sequence of base numbers 10 to 24,        the base sequence of base numbers 9 to 24, the base sequence of        base numbers 8 to 24, the base sequence of base numbers 7 to 24,        the base sequence of base numbers 6 to 24, the base sequence of        base numbers 5 to 24, the base sequence of base numbers 4 to 24,        the base sequence of base numbers 3 to 24, the base sequence of        base numbers 2 to 24 or the base sequence of base numbers 1 to        24 in SEQ ID NO:3.-   13. The following primer (7) or (8):    -   (7) A primer consisting of the base sequence of base numbers 24        to 33, the base sequence of base numbers 23 to 33, the base        sequence of base numbers 22 to 33, the base sequence of base        numbers 21 to 33, the base sequence of base numbers 20 to 33,        the base sequence of base numbers 19 to 33, the base sequence of        base numbers 18 to 33, the base sequence of base numbers 17 to        33, the base sequence of base numbers 16 to 33, the base        sequence of base numbers 15 to 33, the base sequence of base        numbers 14 to 33, the base sequence of base numbers 13 to 33,        the base sequence of base numbers 12 to 33, the base sequence of        base numbers 11 to 33, the base sequence of base numbers 10 to        33, the base sequence of base numbers 9 to 33, the base sequence        of base numbers 8 to 33, the base sequence of base numbers 7 to        33, the base sequence of base numbers 6 to 33, the base sequence        of base numbers 5 to 33, the base sequence of base numbers 4 to        33, the base sequence of base numbers 3 to 33, the base sequence        of base numbers 2 to 33 or the base sequence of base numbers 1        to 33 in SEQ ID NO:4;    -   (8) A primer which is a DNA comprising at most 40 bases and        containing, on the 3 terminal side thereof, the base sequence of        base numbers 24 to 33, the base sequence of base numbers 23 to        33, the base sequence of base numbers 22 to 33, the base        sequence of base numbers 21 to 33, the base sequence of base        numbers 20 to 33, the base sequence of base numbers 19 to 33,        the base sequence of base numbers 18 to 33, the base sequence of        base numbers 17 to 33, the base sequence of base numbers 16 to        33, the base sequence of base numbers 15 to 33, the base        sequence of base numbers 14 to 33, the base sequence of base        numbers 13 to 33, the base sequence of base numbers 12 to 33,        the base sequence of base numbers 11 to 33, the base sequence of        base numbers 10 to 33, the base sequence of base numbers 9 to        33, the base sequence of base numbers 8 to 33, the base sequence        of base numbers 7 to 33, the base sequence of base numbers 6 to        33, the base sequence of base numbers 5 to 33, the base sequence        of base numbers 4 to 33, the base sequence of base numbers 3 to        33, the base sequence of base numbers 2 to 33 or the base        sequence of base numbers 1 to 33 in SEQ ID NO:4.-   14. A primer set comprising a primer according to Item 10 and a    primer according to Item 11.-   15. A primer set comprising a primer according to Item 12 and a    primer according to Item 13.-   16. A PCR kit comprising a primer set according to Item 14 and a    primer set according to Item 15.-   17. A method for detecting bacteria which comprises the step i) of    extracting DNAs contained in a foodstuff sample to be tested    therefrom and the step ii) of carrying out the PCR using a primer    according to any of Items 1 to 13 with the DNA extracted as template    and detecting the DNA thus amplified.-   18. A method for detecting bacteria which comprises the step i) of    extracting DNAs contained in a foodstuff sample to be tested    therefrom and the step ii) of carrying out the PCR using a primer    set according to Item 14 or 15 with the DNA extracted as template    and detecting the DNA thus amplified.-   19. A method for detecting bacteria according to Item 17 or 18,    wherein the DNA extracting step i) comprises the step a) of grinding    the foodstuff sample to be tested, the step b) of centrifuging the    ground sample by a centrifugal force of 50 to 300×g, the step c) of    centrifuging the supernatant obtained by centrifugation in step b)    by a centrifugal force of 2000 to 14,000×g and the step d) of    extracting DNAs contained in the sediment obtained by centrifugation    in step c) therefrom.-   20. A primer which, when used in PCR under appropriate conditions,    serves to detectably amplify 16S rRNA-encoding DNAs of bacteria of    the Enterobacteriaceae and Vibrioaceae families, but when used in    PCR under the same conditions, does not serve to detectably amplify    either chloroplast 16S rRNA-encoding DNAs or mitochondrial 16S    rRNA-encoding DNAs.-   21. A primer which, when used in PCR under appropriate conditions,    serves to detectably amplify 16S rRNA-encoding DNAs of bacteria of    the Staphylococcaceae and Bacillaceae families, but when used in PCR    under the same conditions, does not serve to detectably amplify    either chloroplast 16S rRNA-encoding DNAs or mitochondrial 16S    rRNA-encoding DNAs.

DETAILED DESCRIPTION OF THE INVENTION

(I) Primers

(I-I) Basic Constitution of Primers

The first primer of the invention is a primer which, when used in PCRunder appropriate conditions, serves to detectably amplify 16S r-DNAs ofbacteria of the Escherichia, Salmonella and Vibrio genera, but does notserve to detectably amplify either chloroplast 16S r-DNAs ormitochondrial 16S r-DNAs, when used in PCR under the same conditions.

The second primer of the invention is a primer which, when used in PCRunder appropriate conditions, serves to detectably amplify 16S r-DNAs ofStaphylococcus aureus and Bacillus cereus, but when used in PCR underthe same conditions, does not serve to detectably amplify eitherchloroplast 16S r-DNAs or mitochondrial 16S r-DNAs.

The appropriate conditions mentioned above include those conditionsgenerally employed in PCR procedures, for example, the denaturationcarried out at about 90-98° C. for about 3 seconds to 1 minute, theannealing at about 50-65° C. for about 5 seconds to 2 minutes, the DNAelongation at about 60-75° C. for about 10 seconds to 3 minutes, andabout 1-3.5 mM of the Mg ion concentration.

When used in PCR under appropriate conditions, the first primer of theinvention serves to detectably amplify 16S r-DNAs of bacteria of theEscherichia, Salmonella and Vibrio genera and may serve to detectablyamplify 16S r-DNAs of bacteria of the Enterobacteriaceae and Vibrioaceaefamilies. It is preferable that, under the same conditions, the aboveprimer does not serve to detectably amplify any of the DNAs derived frombacteria not belonging to the Enterobacteriaceae or Vibrioaceae family,algae, fungi, protist, etc. It is preferable that the above primer doesnot serve to detectably amplify 16S r-DNAs of bacteria not belonging tothe Enterobacteriaceae or Vibrioaceae family, in particular. Thus,preferably, the first primer of the invention, when used in PCR underappropriate conditions, serves to detectably amplify 16S r-DNAs ofbacteria of the Escherichia, Salmonella and Vibrio genera, but when usedin PCR under the same conditions, does not serve to detectably amplify16S r-DNAs of bacteria other than the group of bacteria consisting ofbacteria of the Enterobacteriaceae and Vibrioaceae families. By sayingthat the primer does not serve to detectably amplify 16S r-DNAs ofbacteria other than the group of bacteria consisting of bacteria of theEnterobacteriaceae and Vibrioaceae families, it is not only meant thatnone of those other bacteria is detected but also meant that cases whereone to several other bacterial species are exceptionally detected areincluded.

The bacteria of the family Enterobacteriaceae include bacteria of theEscherichia, Salmonella, Shigella, Citrobacter, Klebsiella,Enterobacter, Erwinia, Serratia, Hafnia, Edwardsiella, Yersinia,Providencia genera, etc. The bacteria of the family Vibrioaceae includebacteria of the Vibrio, Photobacterium, Listonella genera, etc. In thisinvention, bacteria of the genus Aeromonas are included in bacteria ofthe Vibrioaceae family.

The bacteria other than the group of bacteria consisting of bacteria ofthe Enterobacteriaceae and Vibrioaceae families are not particularlyrestricted.

The second primer of the invention, when used in PCR under appropriateconditions, serves to detect 16S r-DNAs of Staphylococcus aureus andBacillus cereus and may serve to detectably amplify 16S r-DNAs ofbacteria of the Staphylococcaceae and Bacillaceae families. Preferably,the second primer of the invention serves to detect 16S r-DNAs ofbacteria of the Staphylococcus and Bacillus genera.

Preferably, the second primer of the invention does not serve todetectably amplify any of the DNAs derived from bacteria other than thegroup of bacteria consisting of bacteria of the Staphylococcaceae,Bacillaceae and Aerococcaceae families, algae, fungi, protist and soforth under the same conditions. Preferably, in particular, it does notserve to detectably amplify 16S r-DNAs of bacteria other than the groupof bacteria consisting of bacteria of the Staphylococcaceae, Bacillaceaeand Aerococcaceae families. Thus, preferably, the second primer of theinvention serves to detectably amplify 16S r-DNAs of Staphylococcusaureus and Bacillus cereus when used in PCR under appropriateconditions, but when used in PCR under the same conditions, does notserve to detectably amplify 16S r-DNAs of bacteria other than the groupof bacteria consisting of bacteria of the Staphylococcaceae, Bacillaceaeand Aerococcaceae families. By saying that the above primer does notserve to detectably amplify 16S r-DNAs of bacteria other than the groupof bacteria consisting of bacteria of the Staphylococcaceae, Bacillaceaeand Aerococcaceae families, it is not only meant that none of thoseother bacteria is detected but also meant that cases where one toseveral other bacterial species are exceptionally detected are included.

The bacteria of the family Staphylococcaceae include bacteria of theStaphylococcus genus. The bacteria of the Staphylococcus genus includesuch species as Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus xylosus, and Staphylococcus succinus. The bacteria of theBacillaceae family include bacteria of the Bacillus genus. The bacteriaof the Bacillus genus include Bacillus cereus, Bacillus thuringiensis,Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillussubtilis, etc. The bacteria of the Aerococcaceae family include bacteriaof the Aerococcus genus. The bacteria of the Aerococcus genus includeAerococcus viridans, Aerococcus urinae, etc.

The bacteria other than the group of bacteria consisting of bacteria ofthe Staphylococcaceae, Bacillaceae and Aerococcaceae families are notparticularly restricted.

(I-II) Significance of the Primers of the Invention

The reason why the first primer of the invention is intended fordetecting bacteria of the Escherichia, Salmonella and Vibrio genera andthe second primer of the invention is intended for detecting bacteria ofthe Staphylococcus (in particular Staphylococcus aureus) and Bacillus(in particular Bacillus cereus) genera is as follows.

Thus, bacteria of the Escherichia genus are bacteria regarded as anindicator of oral contamination, and the possibility of the main speciesthereof, namely Escherichia coli, being widely found in foodstuffs ishigh. Recently, highly lethal strains, such as the pathogenicEscherichia coli O-157, have also been found. Bacteria of the Salmonellagenus are mostly found in eggs, fishes and shellfishes and, in Japan,they are food poisoning causing bacteria most frequently reported.Bacteria of the Vibrio genus are detected in marine fishes andshellfishes. Further, Staphylococcus aureus belonging to theStaphylococcus genus is widely detected in foodstuffs as a result ofstaining or contamination of fingers and so forth. Further, Bacilluscereus belonging to the Bacillus genus is relatively often detected inrice and noodle products. Thus, bacteria of the Escherichia, Salmonella,and Vibrio genera, Staphylococcus aureus and Bacillus cereus are theimportant food poisoning causing bacteria reported in Japan.

Further, in some countries, testing for Escherichia coli, bacteria ofthe Salmonella and Vibrio genera and Staphylococcus aureus is legallyobligatory. Bacillus cereus is a bacterial species for which testing isconducted voluntarily by many foodstuff manufacturers.

(I-III) Target Foodstuffs

The foodstuffs which are the targets of the PCR using the primer(s) ofthe present invention are not particularly restricted but widelyinclude, among others, noodle products, meats, fishes, shellfishes,vegetables, cereals, dairy products, and products resulting fromprocessing of these.

(I-IV) Specific Examples of the First Primer

The base sequence of the primer of the invention for detecting bacteriaincludes base sequences complementary to the base sequence of the DNA tobe detected, and base sequences derived from such complementary basesequences by deletion, insertion or substitution of about 1 to 5 bases.

The base sequence of the primer of the invention for detecting bacteriafurther includes the base sequences described in the presentspecification, base sequences derived therefrom by deletion, insertionor substitution of about 1 to 5 bases, and base sequences complementarythereto.

Typically, the first primer of the invention may be the following primerA) or B).

-   A) The following primer (1) or (2):-   (1) A primer comprising the base sequence of base numbers 21 to 30,    the base sequence of base numbers 20 to 30, the base sequence of    base numbers 19 to 30, the base sequence of base numbers 18 to 30,    the base sequence of base numbers 17 to 30, the base sequence of    base numbers 16 to 30, the base sequence of base numbers 15 to 30,    the base sequence of base numbers 14 to 30, the base sequence of    base numbers 13 to 30, the base sequence of base numbers 12 to 30,    the base sequence of base numbers 11 to 30, the base sequence of    base numbers 10 to 30, the base sequence of base numbers 9 to 30,    the base sequence of base numbers 8 to 30, the base sequence of base    numbers 7 to 30, the base sequence of base numbers 6 to 30, the base    sequence of base numbers 5 to 30, the base sequence of base numbers    4 to 30, the base sequence of base numbers 3 to 30, the base    sequence of base numbers 2 to 30 or the base sequence of base    numbers 1 to 30 in the sequence identification number 1 (SEQ    ID-NO:1);-   (2) A primer which is a DNA comprising at most 40 bases and    containing, on the 3 terminal side thereof, the base sequence of    base numbers 21 to 30, the base sequence of base numbers 20 to 30,    the base sequence of base numbers 19 to 30, the base sequence of    base numbers 18 to 30, the base sequence of base numbers 17 to 30,    the base sequence of base numbers 16 to 30, the base sequence of    base numbers 15 to 30, the base sequence of base numbers 14 to 30,    the base sequence of base numbers 13 to 30, the base sequence of    base numbers 12 to 30, the base sequence of base numbers 11 to 30,    the base sequence of base numbers 10 to 30, the base sequence of    base numbers 9 to 30, the base sequence of base numbers 8 to 30, the    base sequence of base numbers 7 to 30, the base sequence of base    numbers 6 to 30, the base sequence of base numbers 5 to 30, the base    sequence of base numbers 4 to 30, the base sequence of base numbers    3 to 30, the base sequence of base numbers 2 to 30 or the base    sequence of base numbers 1 to 30 in SEQ ID NO:1.

Thus, the primer A) is a DNA having any of the base sequences in SEQ IDNO:1 as enumerated above or a DNA having a length of at most 40 bases asa whole and resulting from addition of an arbitrary base sequence to the5 terminal side of any of the base sequences enumerated above.

Preferred among them is a primer consisting of the base sequence of basenumbers 16 to 30, the base sequence of base numbers 15 to 30, the basesequence of base numbers 14 to 30, the base sequence of base numbers 13to 30, the base sequence of base numbers 12 to 30, the base sequence ofbase numbers 11 to 30, the base sequence of base numbers 10 to 30, thebase sequence of base numbers 9 to 30, the base sequence of base numbers8 to 30, the base sequence of base numbers 7 to 30, the base sequence ofbase numbers 6 to 30, the base sequence of base numbers 5 to 30, thebase sequence of base numbers 4 to 30, the base sequence of base numbers3 to 30, the base sequence of base numbers 2 to 30 or the base sequenceof base numbers 1 to 30 in SEQ ID NO:1, or a primer which is a DNAcomprising at most 40 bases and containing any of these base sequenceson the 3 terminal side thereof.

More preferred is a primer consisting of the base sequence of basenumbers 11 to 30, the base sequence of base numbers 10 to 30, the basesequence of base numbers 9 to 30, the base sequence of base numbers 8 to30, the base sequence of base numbers 7 to 30, the base sequence of basenumbers 6 to 30, the base sequence of base numbers 5 to 30, the basesequence of base numbers 4 to 30, the base sequence of base numbers 3 to30, the base sequence of base numbers 2 to 30 or the base sequence ofbase numbers 1 to 30 in SEQ ID NO:1, or a primer which is a DNAcomprising at most 40 bases and containing any of these base sequenceson the 3 terminal side thereof.

Still more preferred is a primer consisting of the base sequence of basenumbers 6 to 30, the base sequence of base numbers 5 to 30, the basesequence of base numbers 4 to 30, the base sequence of base numbers 3 to30, the base sequence of base numbers 2 to 30 or the base sequence ofbase numbers 1 to 30 in SEQ ID NO:1, or a primer which is a DNAcomprising at most 40 bases and containing any of these base sequenceson the 3 terminal side thereof.

Most preferred is the primer consisting of the base sequence of basenumbers 1 to 30 in SEQ ID NO:1, or a primer which is a DNA comprising atmost 40 bases and containing this base sequence on the 3 terminal sidethereof.

-   B) The following primer (3) or (4):-   (3) A primer consisting of the base sequence of base numbers 14 to    23, the base sequence of base numbers 13 to 23, the base sequence of    base numbers 12 to 23, the base sequence of base numbers 11 to 23,    the base sequence of base numbers 10 to 23, the base sequence of    base numbers 9 to 23, the base sequence of base numbers 8 to 23, the    base sequence of base numbers 7 to 23, the base sequence of base    numbers 6 to 23, the base sequence of base numbers 5 to 23, the base    sequence of base numbers 4 to 23, the base sequence of base numbers    3 to 23, the base sequence of base numbers 2 to 23 or the base    sequence of base numbers 1 to 23 in SEQ ID NO:2;-   (4) A primer which is a DNA comprising at most 40 bases and    containing, on the 3 terminal side thereof, the base sequence of    base numbers 14 to 23, the base sequence of base numbers 13 to 23,    the base sequence of base numbers 12 to 23, the base sequence of    base numbers 11 to 23, the base sequence of base numbers 10 to 23,    the base sequence of base numbers 9 to 23, the base sequence of base    numbers 8 to 23, the base sequence of base numbers 7 to 23, the base    sequence of base numbers 6 to 23, the base sequence of base numbers    5 to 23, the base sequence of base numbers 4 to 23, the base    sequence of base numbers 3 to 23, the base sequence of base numbers    2 to 23 or the base sequence of base numbers 1 to 23 in SEQ ID NO:2.

Thus, the primer B) is a DNA having any of the base sequences in SEQ IDNO:2 as enumerated above or a DNA having a length of at most 40 bases asa whole and resulting from addition of an arbitrary base sequence to the5 terminal side of any of the base sequences enumerated above.

Preferred among them is a primer consisting of the base sequence of basenumbers 9 to 23, the base sequence of base numbers 8 to 23, the basesequence of base numbers 7 to 23, the base sequence of base numbers 6 to23, the base sequence of base numbers 5 to 23, the base sequence of basenumbers 4 to 23, the base sequence of base numbers 3 to 23, the basesequence of base numbers 2 to 23 or the base sequence of base numbers 1to 23 in SEQ ID NO:2, or a primer which is a DNA comprising at most 40bases and containing any of these base sequences on the 3 terminal sidethereof.

More preferred is a primer consisting of the base sequence of basenumbers 4 to 23, the base sequence of base numbers 3 to 23, the basesequence of base numbers 2 to 23 or the base sequence of base numbers 1to 23 in SEQ ID NO:2, or a primer which is a DNA comprising at most 40bases and containing any of these base sequences on the 3 terminal sidethereof. Most preferred is the primer consisting of the base sequence ofbases 1 to 23 in SEQ ID NO:2, or a primer which is a DNA comprising atmost 40 bases and containing this base sequence on the 3 terminal sidethereof.

In the primers A) and B), the base sequence optionally added on the 5terminal side preferably comprises about 1 to 10 bases, in particularabout 1 to 5 bases. Most preferably, however, no addition is made ofsuch optional base sequence.

(I-V) Specific Examples of the Second Primer

Typically, the second primer of the invention may be the followingprimer C) or D).

-   C) The following primer (5) or (6):-   (5) A primer consisting of the base sequence of base numbers 15 to    24, the base sequence of base numbers 14 to 24, the base sequence of    base numbers 13 to 24, the base sequence of base numbers 12 to 24,    the base sequence of base numbers 11 to 24, the base sequence of    base numbers 10 to 24, the base sequence of base numbers 9 to 24,    the base sequence of base numbers 8 to 24, the base sequence of base    numbers 7 to 24, the base sequence of base numbers 6 to 24, the base    sequence of base numbers 5 to 24, the base sequence of base numbers    4 to 24, the base sequence of base numbers 3 to 24, the base    sequence of base numbers 2 to 24 or the base sequence of base    numbers 1 to 24 in SEQ ID NO:3;-   (6) A primer which is DNA comprising at most 40 bases and    containing, on the 3 terminal side thereof, the base sequence of    base numbers 15 to 24, the base sequence of base numbers 14 to 24,    the base sequence of base numbers 13 to 24, the base sequence of    base numbers 12 to 24, the base sequence of base numbers 11 to 24,    the base sequence of base numbers 10 to 24, the base sequence of    base numbers 9 to 24, the base sequence of base numbers 8 to 24, the    base sequence of base numbers 7 to 24, the base sequence of base    numbers 6 to 24, the base sequence of base numbers 5 to 24, the base    sequence of base numbers 4 to 24, the base sequence of base numbers    3 to 24, the base sequence of base numbers 2 to 24 or the base    sequence of base numbers 1 to 24 in SEQ ID NO:3.

Thus, the primer C) is a DNA having any of the base sequences in SEQ IDNO:3 as enumerated above or a DNA having a length of at most 40 bases asa whole and resulting from addition of an arbitrary base sequence to the5 terminal side of any of the base sequences enumerated above.

Preferred among them is a primer comprising the base sequence of basenumbers 10 to 24, the base sequence of base numbers 9 to 24, the basesequence of base numbers 8 to 24, the base sequence of base numbers 7 to24, the base sequence of base numbers 6 to 24, the base sequence of basenumbers 5 to 24, the base sequence of base numbers 4 to 24, the basesequence of base numbers 3 to 24, the base sequence of base numbers 2 to24 or the base sequence of base numbers 1 to 24 in SEQ ID NO:3, or aprimer which is a DNA comprising at most 40 bases and containing any ofthese base sequences on the 3 terminal side thereof.

More preferred is a primer consisting of the base sequence of basenumbers 5 to 24, the base sequence of base numbers 4 to 24, the basesequence of base numbers 3 to 24, the base sequence of base numbers 2 to24 or the base sequence of base numbers 1 to 24 in SEQ ID NO:3, or aprimer which is a DNA comprising at most 40 bases and containing any ofthese base sequences on the 3 terminal side thereof.

Most preferred is the primer consisting of the base sequence of basenumbers 1 to 24 in SEQ ID NO:3, or a primer which is a DNA comprising atmost 40 bases and containing this base sequence on the 3 terminal sidethereof.

-   D) The following primer (7) or (8):-   (7) A primer consisting of the base sequence of base numbers 24 to    33, the base sequence of base numbers 23 to 33, the base sequence of    base numbers 22 to 33, the base sequence of base numbers 21 to 33,    the base sequence of base numbers 20 to 33, the base sequence of    base numbers 19 to 33, the base sequence of base numbers 18 to 33,    the base sequence of base numbers 17 to 33, the base sequence of    base numbers 16 to 33, the base sequence of base numbers 15 to 33,    the base sequence of base numbers 14 to 33, the base sequence of    base numbers 13 to 33, the base sequence of base numbers 12 to 33,    the base sequence of base numbers 11 to 33, the base sequence of    base numbers 10 to 33, the base sequence of base numbers 9 to 33,    the base sequence of base numbers 8 to 33, the base sequence of base    numbers 7 to 33, the base sequence of base numbers 6 to 33, the base    sequence of base numbers 5 to 33, the base sequence of base numbers    4 to 33, the base sequence of base numbers 3 to 33, the base    sequence of base numbers 2 to 33 or the base sequence of base    numbers 1 to 33 in SEQ ID NO:4;-   (8) A primer which is a DNA comprising at most 40 bases and    containing, on the 3 terminal side thereof, the base sequence of    base numbers 24 to 33, the base sequence of base numbers 23 to 33,    the base sequence of base numbers 22 to 33, the base sequence of    base numbers 21 to 33, the base sequence of base numbers 20 to 33,    the base sequence of base numbers 19 to 33, the base sequence of    base numbers 18 to 33, the base sequence of base numbers 17 to 33,    the base sequence of base numbers 16 to 33, the base sequence of    base numbers 15 to 33, the base sequence of base numbers 14 to 33,    the base sequence of base numbers 13 to 33, the base sequence of    base numbers 12 to 33, the base sequence of base numbers 11 to 33,    the base sequence of base numbers 10 to 33, the base sequence of    base numbers 9 to 33, the base sequence of base numbers 8 to 33, the    base sequence of base numbers 7 to 33, the base sequence of base    numbers 6 to 33, the base sequence of base numbers 5 to 33, the base    sequence of base numbers 4 to 33, the base sequence of base numbers    3 to 33, the base sequence of base numbers 2 to 33 or the base    sequence of base numbers 1 to 33 in SEQ ID NO:4.

Thus, the primer D) is a DNA having any of the base sequences in SEQ IDNO:4 as enumerated above or a DNA having a length of at most 40 bases asa whole and resulting from addition of an arbitrary base sequence to the5 terminal side of any of the base sequences enumerated above.

Preferred among them is a primer consisting of the base sequence of basenumbers 19 to 33, the base sequence of base numbers 18 to 33, the basesequence of base numbers 17 to 33, the base sequence of base numbers 16to 33, the base sequence of base numbers 15 to 33, the base sequence ofbase numbers 14 to 33, the base sequence of base numbers 13 to 33, thebase sequence of base numbers 12 to 33, the base sequence of basenumbers 11 to 33, the base sequence of base numbers 10 to 33, the basesequence of base numbers 9 to 33, the base sequence of base numbers 8 to33, the base sequence of base numbers 7 to 33, the base sequence of basenumbers 6 to 33, the base sequence of base numbers 5 to 33, the basesequence of base numbers 4 to 33, the base sequence of base numbers 3 to33, the base sequence of base numbers 2 to 33 or the base sequence ofbase numbers 1 to 33 in SEQ ID NO:4, or a primer which is a DNAcomprising at most 40 bases and containing any of these base sequenceson the 3 terminal side thereof.

More preferred is a primer consisting of the base sequence of basenumbers 14 to 33, the base sequence of base numbers 13 to 33, the basesequence of base numbers 12 to 33, the base sequence of base numbers 11to 33, the base sequence of base numbers 10 to 33, the base sequence ofbase numbers 9 to 33, the base sequence of base numbers 8 to 33, thebase sequence of base numbers 7 to 33, the base sequence of base numbers6 to 33, the base sequence of base numbers 5 to 33, the base sequence ofbase numbers 4 to 33, the base sequence of base numbers 3 to 33, thebase sequence of base numbers 2 to 33 or the base sequence of basenumbers 1 to 33 in SEQ ID NO:4, or a primer which is a DNA comprising atmost 40 bases and containing any of these base sequences on the 3terminal side thereof.

Still more preferred is a primer consisting of the base sequence of basenumbers 9 to 33, the base sequence of base numbers 8 to 33, the basesequence of base numbers 7 to 33, the base sequence of base numbers 6 to33, the base sequence of base numbers 5 to 33, the base sequence of basenumbers 4 to 33, the base sequence of base numbers 3 to 33, the basesequence of base numbers 2 to 33 or the base sequence of base numbers 1to 33 in SEQ ID NO:4, or a primer which is a DNA comprising at most 40bases and containing any of these base sequences on the 3 terminal sidethereof.

Most preferred is the primer consisting of the base sequence of basenumbers 1 to 33 in SEQ ID NO:4, or a primer which is a DNA comprising atmost 40 bases and containing this base sequence on the 3 terminal sidethereof.

In the primers C) and D), the base sequence optionally added on the 5terminal side preferably comprises about 1 to 7 bases, in particularabout 1 to 5 bases. Most preferably, however, no addition is made ofsuch optional base sequence.

(I-VI) Method for Producing the Primers

The primers of the invention can be produced with ease by chemicalsynthesis.

(I-VII) Method for Using the Primers

The first primer of the invention can be used in PCR in combination witha known universal primer and capable of detecting eubacteria, or a setof two first primers of the invention can be used as a senseprimer/antisense primer set. In particular, the use of a set of twofirst primers of the invention is preferred. Such use further increasesthe sensitivity in detecting the targeted bacterial group. For example,the primer A) or B) may be used in combination with a primer with whicheubacteria can be detected, or the primer A) and primer B) may be usedin combination as a primer set consisting of a sense primer and anantisense primer. The latter case is more preferred.

The first primer of the invention can be used in detecting bacteria ofthe Escherichia, Salmonella and Vibrio genera. It can be used indetecting bacteria of the Enterobacteriaceae and Vibrioaceae families aswell.

Similarly, the second primer of the invention can be used in PCR incombination with an eubacteria-detectable primer known in the art, or aset of two second primers of the invention can be used as a senseprimer/antisense primer set. In particular, the use of a set of twosecond primers of the invention is preferred. Such use further increasesthe sensitivity in detecting the targeted group of bacteria. Forexample, the primer C) or D) may be used in combination with a primerwith which bacteria in general can be detected, or the primer C) andprimer D) may be used in combination as a primer set consisting of asense primer and an antisense primer. The latter case is more preferred.

The second primer of the invention can be used in detecting bacteria ofthe Staphylococcus (in particular Staphylococcus aureus) and Bacillus(in particular Bacillus cereus) genera.

Furthermore, the first primer set and second primer set of the inventioncan be used in combination as a PCR set. The PCR set of the inventioncan be used in detecting bacteria of the Escherichia, Salmonella andVibrio genera (or bacteria of the Enterobacteriaceae and Vibrioaceaefamilies) as well as bacteria of the Staphylococcus (in particularStaphylococcus aureus) and Bacillus (in particular Bacillus cereus)genera.

More specifically, for testing the presence or absence of a specificbacterial group in foodstuffs using the PCR set of the invention, thePCR using the first primer of the invention to detect bacteria of theEscherichia, Salmonella and Vibrio genera and the PCR using the secondprimer of the invention to detect bacteria of the Staphylococcus (inparticular Staphylococcus aureus) and Bacillus (in particular Bacilluscereus) genera can be carried out separately. By carrying out these twoPCRs simultaneously, it becomes possible to detect the specific targetedbacterial group by spending time and effort only once.

(II) Method for Detacting Bacteria

The method for detecting bacteria according to the invention comprisesthe step i) of extracting DNAs contained in a foodstuff sample to betested therefrom and the step ii) of carrying out the PCR using theprimer of the invention with the DNA extracted as template, followed bydetecting the amplified-DNA.

(II-I) DNA Extraction Step

Target Foodstuffs

The foodstuffs as the targets of the method of the invention are notparticularly restricted. The method of the invention can be applied to awide variety of foodstuffs, including noodle products, meats, fishes,shellfishes, vegetables, cereals, dairy products, and products resultingfrom processing of these, and the like.

Outline of the DNA Extraction Step

In carrying out the method of the invention, the DNA may be extractedeither from a culture of bacteria separated from a foodstuff, ordirectly from the foodstuff. It is also possible to extract the DNA froma fraction possibly containing bacteria after separation of the fractionpossibly containing bacteria from the foodstuff by centrifugation of afoodstuff possibly containing bacteria. The last-mentioned method ismost preferred as the step of DNA extraction from bacteria possiblycontained in the foodstuff.

Preferred DNA Extraction Step

In the following, a preferred DNA extraction step is described indetail. Thus, in the method for detecting bacteria according to theinvention, the DNA extracting step preferably comprises the step a) ofgrinding the foodstuff sample to be tested, the step b) of centrifugingthe ground sample by a centrifugal force of about 50 to 300×g, the stepc) of centrifuging the supernatant obtained by centrifugation in step b)by a centrifugal force of about 2000 to 14,000×g and the step d) ofextracting the DNA contained in the pellets obtained by centrifugationin step c) therefrom. In the following, the respective steps aredescribed in detail.

<Grinding Step>

The foodstuff sample can be ground in the usual manner by adding wateror a buffer solution in an amount of about 5 to 20 times the weight ofthe foodstuff sample and grinding the foodstuff using a Stomacher® orlike homogenizer. In the case of a liquid or semi-liquid foodstuffsample, if those samples are mixed and diluted with water or buffersolution, the grinding step may be omitted.

<Centrifugation Step>

Then, the ground foodstuff sample can be centrifuged generally at about50 to 300×g, preferably at about 80 to 150×g, for about 1 to 2 minutes.This causes the ground foodstuff sample mostly to settle, while thebacteria remain in the supernatant. The supernatant is thereforecollected.

The supernatant can be centrifuged generally at about 2,000 to 14,000×g,preferably at about 8,000 to 14,000×g, for about 2 to 5 minutes. Most ofthe bacteria can be precipitated from the supernatant by thecentrifugation.

Then, to the precipitate is resuspended in water or a buffer solution inan amount of about 1/10 to ½ of the volume of the ground foodstuffliquid subjected to centrifugation treatment, and the resultingsuspension containing bacteria can now be subjected to the DNAextraction step.

DNA Extraction Method

DNA extraction from the foodstuff sample or separated bacteria can becarried out employing any of the methods known in the art. Such knownmethods include the Marmur method, the enzyme method which is amodification of the Marmur method, the benzyl chloride method, and thelike.

For confirming the presence or absence of specific bacteria includingbacteria causing food poisoning prior to shipment of food products, itis necessary to rapidly extract the DNA. An example of the method ofsuch rapid DNA extraction from bacteria is the Chelex method(Biotechniques, April 1991; 10(4):506-513; Microbiol. Res. May 1999;154(1):23-26). The Chelex method comprises adding Chelex® solution in anamount of about 10% by weight to the bacterial suspension, heating on aboiling water bath for about 5 minutes for lysing the bacteria,centrifuging the bacterial lysate, and collecting the supernatant. Thesupernatant contains the DNA, and this DNA extract can be used in PCR.

As another example of the rapid DNA extraction method is the methodcomprising dissolving the bacterial pellets by adding about 10 to 30% byweight of sodium dodecyl sulfate, heating the mixture at about 60-100°C. for about 5-20 minutes for bacterial cell lysis, adding sodiumchloride to the resulting solution to a salt concentration of 1 M,carrying out deproteinization, and subjecting the supernatant to ethanolprecipitation for DNA preparation.

(II-II) Step of DNA Detection by PCR

In carrying out the PCR, the primers of the invention as described aboveare used. Generally, for one foodstuff sample, the PCR using the firstprimer of the invention for detecting bacteria of the Escherichia,Salmonella and Vibrio genera, and the PCR using the second primer of theinvention for detecting bacteria of the Staphylococcus (in particularStaphylococcus aureus) and Bacillus (in particular Bacillus cereus)genera may be carried out.

More preferably, one set of the first primers of the invention are usedas a sense primer and an antisense primer, and one set of the secondprimers of the invention are used as a sense primer and an antisenseprimer. The combined use of a primer A) and a primer B) and the combineduse of a primer C) and a primer D) are particularly preferred.

The PCR conditions are not particularly restricted but optimalconditions may be selected for each PCR apparatus. For example, thefollowing conditions may be used:

-   -   Thermal denaturation of double-stranded DNA to single-stranded        DNA: Heating is generally made at about 90-98° C., preferably at        about 92-96° C., generally for about 3 seconds to 1 minute,        preferably for about 30 seconds to 1 minute.    -   Annealing: Heating is made generally at about 50-65° C.,        preferably at 50-60° C., generally for about 5 seconds to 2        minutes, preferably for about 30 seconds to 1 minute.    -   DNA elongation reaction: Heating is made generally at about        60-75° C., preferably at about 70-74° C., generally for about 10        seconds to 3 minutes, preferably for about 30 seconds to 2        minutes.    -   Mg ion concentration in the reaction liquid: Generally about        1-3.5 mM, preferably about 2-3 mM.

This reaction is carried out generally in about 20-40 cycles, preferablyin about 30 cycles, whereby the target DNA can be amplified to adetectable level.

The PCR apparatus, for example, a commercial apparatus of the block typeor capillary type can be used. For rapid DNA amplification, a capillarytype PCR apparatus is preferably used.

The PCR product may further be subjected to separation by agarose gelelectrophoresis, followed by nucleic acid staining with ethidium bromideor SYBR® Green, for instance.

A PCR amplification product DNA band in each PCR, when detected,indicates the existence of bacteria corresponding to the primer used.

When the PCR is carried out using the first primer and/or second primerof the invention and a band is detected in electrophoresis, thepossibility is indicated of bacteria of the Escherichia, Salmonella andVibrio genera existing in the test foodstuff. When the PCR is carriedout using the primer having the base sequence of base numbers 1 to 30 inSEQ ID NO:1 and the primer having the base sequence of base numbers 1 to23 in SEQ ID NO:2, a band corresponding to a DNA fragment of about 420base pairs will generally be detected if bacteria of the Escherichia,Salmonella or Vibrio genus exist.

When the PCR is carried out using the third primer and/or fourth primerof the invention and a band is detected in electrophoresis, thepossibility is indicated of the existence of bacteria of theStaphylococcus (in particular Staphylococcus aureus) and Bacillus (inparticular Bacillus cereus) genera. When the PCR is carried out usingthe primer having the base sequence of base numbers 1 to 24 in SEQ IDNO:3 and the primer having the base sequence of base numbers 1 to 33 inSEQ ID NO:4, a band corresponding to a DNA fragment of about 380 basepairs will generally be detected if bacteria of the Staphylococcus genusincluding Staphylococcus aureus or bacteria of the Bacillus genusincluding Bacillus cereus are present.

It is also possible to carry out the analysis in a short time by using areal time PCR apparatus. In this case, the occurrence or nonoccurrenceof a PCR amplification product can be confirmed by melting curveanalysis following PCR. In cases where the judgment is difficult, suchagarose gel electrophoresis as mentioned above may be carried out forthe analysis.

In cases where a sufficient quantity of DNA cannot be obtained becauseof the number of bacterial cells obtained being excessively small orwhere the result of PCR is considered to be affected by dead bacterialcells, the number of bacterial cells can be increased by adding asterilized liquid medium for bacterial culture to the test sample and bycarrying out cultivation at about 30-37° C. In this way, the DNAdetection sensitivity can be improved, and the influence of dead cellscan be reduced. The liquid medium is not particularly restricted in typebut may suitably be selected from a wide range of universal culturemedia for bacteria, such as brain heart infusion medium, Tryptosoyamedium and the like. In the case of bacteria of the Vibrio genus, it isadvisable that those media supplemented with about 1-3% sodium chrolideor an alkaline peptone medium are used.

When the foodstuff sample contains about 10 viable cells per gramthereof, about 4-8 hours of cultivation is generally sufficient for theDNA thereof to become detectable by the PCR described above. Suchbacterial contamination that the number of bacteria to be detected (inparticular bacteria of the Escherichia genus, Salmonella genus,Staphylococcus aureus or Bacillus cereus) in each gram of a foodstuff isless than 10 can be generally disregarded. This is because when thenumber of bacteria contained in each gram of a foodstuff is less than10, smearing of a plate agar medium with the ground food liquid will notresult in colony detection, hence such contamination is generallyneglected. In cases where it is desired that the detectable number ofbacteria per unit food weight be lowered, the sample size may beincreased and/or the cultivation time may adequately be prolonged.

(III) Effects of the Invention

In accordance with the present invention, there are provided, PCRprimers with which the presence or absence of a specific bacterialgroup, inclusive of food poisoning-causing bacteria, in foodstuffs canbe detected easily and rapidly, and a method for easily and rapidlydetecting the presence or absence of a specific bacterial group infoodstuffs using those primers.

In addition, when the primers of the invention are used, such DNAs asfood-derived chloroplast DNA and mitochondrial DNA are not detected and,therefore, the PCR can be carried out without cultivation of bacteriafollowing separation thereof from foodstuff samples. The time fortesting can be shortened accordingly.

The crucial bacteria as food poisoning-causing bacteria in foodstuffsinclude bacteria of the genus Escherichia, bacteria of the Salmonella,Vibrio, Staphylococcus (in particular Staphylococcus aureus) andBacillus (in particular Bacillus cereus) genera. Under suchcircumstances, the first primer of the invention can serve to detect arelatively broad range group of bacteria including bacteria of theEscherichia, Salmonella and Vibrio genera, while the second primer ofthe invention also can serve to detect a relatively broad range group ofbacteria including Staphylococcus aureus and Bacillus cereus (including,in some instances, bacteria of the Staphylococcus and Bacillus genera).Therefore, when two PCR procedures using these two primers respectivelyare concurrently carried out, it is possible to expediently check thelikelihood of contamination by main food poisoning causative bacteria.

Therefore, the primers of the invention can be preferably used inprimary screening of bacterial testing of foodstuffs with a relativelylow frequency of contamination. Thus, when no bacteria are detected inthe primary screening using the primers of the invention, it issuggested that the above-mentioned group of bacteria detectable withthose primers do not exist. When bacteria are detected in the primaryscreening using the primers of the invention, the bacterial species maybe identified by carrying out the PCR, as a secondary screening, usingprimers specific to specific bacterial species.

When, in carrying out the method of the invention, the food grindingstep and centrifugation procedure are carried out, bacteria can beefficiently separated and collected from foodstuff samples and,therefore, it is no more necessary to separate bacteria from foodstuffsamples and cultivate the same, hence the testing time can be cut downaccordingly.

EXAMPLES

The following examples illustrate the present invention morespecifically. It is to be noted, however, that these examples are neverrestrictive of the scope of the present invention.

Example 1 Confirmation of Reactivity in Type Strains

<DNA Preparation>

Bacterial strains of Escherichia coli JCM 1649^(T) , Salmonellatyphimurium IFO 13245, Staphylococcus aureus IFO 3060 and Bacilluscereus IFO 15305^(T) were grown in brain heart infusion medium. UsingDNeasy Tissue Kit (product of QIAGEN), DNA solutions were prepared fromcells of the respective strains.

<Primer Preparation>

The DNA primers defined by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQID NO:4 were synthesized using a DNA synthesizer.

<PCR>

The DNA amount in cells of each bacterial strain was determined, andtest DNA solutions were prepared so that the genome copy numbercontained in the DNA solutions as estimated from the molecular weight ofthe genome of each bacterial strain might amount to about 10 copies/μl,about 10² copies/μl or about 10³ copies/μl. Sterilized water and thebuffer solution for PCR were added to 1 μl of each test DNA solution foradjustment to the following final concentrations: 2.0 mM MgCl₂, 50 mMTris-HCl (pH 8.3), 0.25 mg/ml BSA, 200 μM dNTPs, 0.2 μM of each primer.Thereto was added 1.0 unit of Hot Start Taq DNA polymerase (product ofRoche Diagnostics), and the PCR was carried out with a total liquidamount of 20 μl.

For Escherichia coli and Salmonella typhimurium, the primers of SEQ IDNO:1 and SEQ ID NO:2 were used in combination. For Staphylococcus aureusand Bacillus cereus, the primers of SEQ ID NO:3 and SEQ ID NO:4 wereused in combination.

As for the temperature conditions for PCR, the reaction mixture wasmaintained at 95° C. for 15 minutes, then thermal denaturation wascarried out at 95° C. for 5 seconds, annealing at 59° C. for 10 secondsand DNA elongation at 72° C. for 20 seconds. Under these conditions, thePCR was carried out in 30 cycles. The PCR apparatus used was LightCycler(product of Roche Diagnostics).

Each PCR product obtained was electrophoresed on 1.6% agarose gel(TaKaRa) at 100 V for 25 minutes using Mupid-2 (Cosmo Bio) and, afterstaining with ethidium bromide (1.0 μg/ml), band observation was madeunder a UV lamp.

The agarose gel electrophoretic patterns obtained are shown in FIG. 1.(A) and (B) in FIG. 1 are the results obtained by using the primers ofSEQ ID NO:1 and SEQ ID NO:2 in combination, and (C) and (D) in FIG. 1are the results obtained by using the primers of SEQ ID NO:3 and SEQ IDNO:4 in combination. (A) in FIG. 1 shows the result with Escherichiacoli, (B) in FIG. 1 the result with Salmonella typhimurium, (C) in FIG.1 the result with Staphylococcus aureus, and (D) in FIG. 1 the resultwith Bacillus cereus.

From FIG. 1, it was demonstrated that the DNAs of Escherichia coli andSalmonella typhimurium can be detected by the combined use of theprimers of SEQ ID NO:1 and SEQ ID NO:2. It was also demonstrated thatthe DNAs of Staphylococcus aureus and Bacillus cereus can be detected bythe combined use of the primers of SEQ ID NO:3 and SEQ ID NO:4. Further,it was confirmed that when such a genomic DNA exists in a quantity ofabout 10² copies, the DNA can be detected under the above-specified PCRconditions.

Example 2 Reactivity with Plant DNAs

<DNA Preparation>

Respective plant DNAs were extracted from commercially available wheatflour, potato, soybean and corn using DNeasy Plant Mini Kit (product ofQiagen).

<PCR>

After determination of the DNA concentration in each plant DNA extractprepared, test DNA solutions corresponding in genomic DNA copy number ineach solution to about 10⁰ copy/μl, about 10¹ copies/μl, about 10²copies/μl and about 10³ copies/μl as estimated from the molecular weightof DNA per genome in each plant species were prepared. Sterilized waterand the buffer solution for PCR were added to 1 μl of each of these testDNA solutions for adjustment to the following final concentrations: 2.0mM MgCl₂, 50 mM Tris-HCl (pH 8.3), 0.25 mg/ml BSA, 200 μM dNTPs, and 0.2μM of each primer. Thereto was added 1.0 unit of Hot Start Taq DNApolymerase (product of Roche Diagnostics), and the PCR was carried outwith a total liquid amount of 20 μl.

As for the primers, a set of the primer of SEQ ID No:1 and the primer ofSEQ ID NO:2, a set of the primer of SEQ ID NO:3 and the primer of SEQ IDNO:4, and a primer set reported to serve to detect bacteria in general(J Dent Res 78(4):850-856, April, 1999) were used respectively.

As for the temperature conditions for PCR, the reaction mixture wasmaintained at 95° C. for 15 minutes, then thermal denaturation wascarried out at 95° C. for 5 seconds, annealing at 59° C. for 10 secondsand DNA elongation at 72° C. for 20 seconds. Under these conditions, thePCR was carried out in 30 cycles. The PCR apparatus used was LightCycler(product of Roche Diagnostics).

Each PCR product obtained was electrophoresed on 1.6% agarose gel(TaKaRa) at 100 V for 25 minutes using Mupid-2 (Cosmo Bio). The gelobtained after electrophoresis was stained with ethidium bromide (1.0μg/ml), and DNA band observation was made under a UV lamp. The resultsare shown in FIG. 2. (A) in FIG. 2 shows the result with wheat, (B) inFIG. 2 with potato, (C) in FIG. 2 with soybean, and (D) in FIG. 2 withcorn.

As shown in FIG. 2, with all of wheat, potato, soybean and corn, no bandwas detected when the primers of SEQ ID NO:1 and SEQ ID NO:2 or theprimers of SEQ ID NO:3 and SEQ ID NO:4 were used. It is thus evidentthat the PCR using the primers of the invention does not detect theseplant-derived DNAs. On the other hand, the use of the above-mentionedprimer set reported to be capable of detecting bacteria in generalresulted in positive band detection. This is probably due to thereaction with plant-derived DNAs, including chloroplast or/andmitochondrial DNAs. The DNA base sequences of these amplification-duebands were examined and found to coincide with the chloroplast 16S rRNAgenes of the respective plants.

Further, the same experiments were carried out using bovine DNA (productof GeneScan), chicken DNA (product of GeneScan), salmon DNA (product ofClontech), Saccharomyces cerevisiae IFO 0282 DNA and Aspergillus oryzaeIFO 4206 DNA. It was confirmed that when the primers of SEQ ID NO:1 andSEQ ID NO:2 or the primers of SEQ ID NO:3 and SEQ ID NO:4 were used, noband was detected with any of the DNAS. It was thus found that thoseDNAs derived from mammals, birds, fishes and fungi are not detected bythe PCR using the primers of the invention.

Example 3 Confirmation of Species Specificity of the Primers of theInvention

The bacterial strains listed below in Table 1 were cultured in the samemanner as in Example 1 using media suited for the strains, and the DNAwas extracted from the culture of each strain and subjected to PCR. Theprimers used were the primer of SEQ ID NO:1 in combination with theprimer of SEQ ID NO:2, and the primer of SEQ ID NO:3 in combination withthe primer of SEQ ID NO:4.

The results are shown in Table 1.

TABLE 1 Primers of Primers of SEQ ID SEQ ID Nos.: family Genus, Species,and strains Nos.: 1 and 2 3 and 4 Rhizobiaceae Agrobacterium radiobacterATCC 19358^(T) − − Alcaligenaceae Alcaligenes faecalis JCM 1474^(T) − −Neisseriaceae Chromobacterium violaceum JCM 1249^(T) − − Neisseriameningitides ATCC 13077^(T) − − Xanthomonadaceae Xanthomonas maltophiliaJCM 1975^(T) − − Legionellaceae Legionella pneumophila JCM 7571^(T) − −Pseudomonadaceae Pseudomonas aeruginosa ATCC 27843^(T) − − Pseudomonasfluorescens JCM 5963^(T) − − Moraxellaceae Moraxella antipestifer JCM9532^(T) − − Acinetobacter baumannii JCM 6841^(T) − − VibrionaceaeVibrio vulnificus JCM 3725^(T) + − Vibrio parahaemolyticus ATCC17802^(T) + − Photobacterium leiognathi ATCC 25521^(T) + − Aeromonashydrophila ATCC 7966^(T) (1) + − Enterobacteriaceae Enterobacteragglomerans JCM 1236^(T) + − Enterobacter cloacae JCM 1232^(T) + −Citrobacter freundii JCM 1657^(T) + − Escherichia coli JCM 1649^(T) + −Escherichia coli JM109 + − Escherichia coli HB101 + − Escherichia coli0157: H7 ATCC 43889 + − Hafnia alvei JCM 1666^(T) + − Klebsiellapneumoniae JCM 1662^(T) + − Plesiomonas shigelloides ATCC 14029^(T) + −Proteus mirabilis JCM 1669^(T) − − Salmonella typhimurium IFO 13245 + −Salmonella typhimurium IFO 14211 + − Salmonella enteritidis IFO 3313 + −Serratia ficaria JCM 1241^(T) + − Serratia marcescens JCM 1239^(T) + −Shigella flexneri ATCC 29903^(T) + − Yersina enterocolitica ATCC9610^(T) + − Pasteurellaceae Haemophilus influenzae ATCC 33391^(T) − −Campylobacteraceae Campylobacter coli ATCC 33559^(T) − −Helicobacteraceae Helicobacter pylori ATCC 43504^(T) − − ClostridiaceaeClostridium perfringens JCM 1290^(T) − − Eubacteriaceae Eubacteriumalactolyticum JCM 6480^(T) (2) − − Acidaminococcaceae Veillonellaalcalescens ATCC 27215 − − Bacillaceae Bacillus cereus IFO 15305^(T) − +Bacillus cereus ATCC 11950 − + Bacillus thuringiensis ATCC 10792^(T) − +Bacillus licheniformis JCM 2505^(T) − + Bacillus megaterium JCM 2506^(T)− + Bacillus pumilus JCM 2508^(T) − + Bacillus subtilis IFO 13719^(T)− + Staphylococcaceae Staphylococcus aureus IFO 3060 − + Staphylococcusaureus JCM 2874 − + Staphylococcus epidermides JCM 2414^(T) − +Lactobacillaceae Lactobacillus casei JCM 1134^(T) − − Lactobacillusgasseri JCM 1131^(T) − − Lactobacillus bulgaricus JCM 1002^(T) − −Aerococcaceae Aerococcus viridans IFO 12219^(T) − + StreptococcaceaeStreptococcus mutans JCM 5705^(T) − − Streptococcus salivarius JCM5707^(T) − − Streptococcus agalactiae JCM 5671^(T) − − CoriobacteriaceaeCollinsella aerofaciens JCM 10188^(T) − − Actinomycetaceae Actinomycesnaeslundii JCM 8349^(T) − − Micrococcaceae Micrococcus luteus JCM1464^(T) − − Microbacteriaceae Microbacterium lacticum JCM 1379 − −Corynebacteriaceae Corynebacterium kutscheri JCM 9385^(T) − −Corynebacterium xerosis JCM 1971^(T) − − PropionibacteriaceaePropionibacterium acnes JCM 6425^(T) − − BifidobacteriaceaeBifidobacterium longum JCM 1217^(T) − − Bifidobacterium adolescentis JCM1275^(T) − − Bacteroidaceae Bacteroides vulgatus JCM 5826^(T) − −Porphyromonadaceae Porphyromonas gingivalis ATCC 33277^(T) − −Prevotellaceae Prevotella intermedia ATCC 25611^(T) − −Flavobacteriaceae Flavobacterium johnsoniae JCM 8514^(T) − −Flexibacteraceae Cytophaga arvensicola JCM 2836^(T) − − FusobacteriaceaeFusobacterium nucleatum ATCC 25586^(T) − − (1): The Aeromonas genus iscurrently belonged to the Vibrioaceae family. Recently, it has beenproposed that it should be reclassified in the new Aeromodaceae family(TAXONOMIC OUTLINE OF THE PROCARYOTE GENERA, BERGEY S MANUAL OFSYSTEMATIC BACTERIOLOGY, SECOND EDITION Release 1.0, April 2001). (2):The genus has been reclassified in Pseudoramibacter alactolyticym (Clin.Infect. Dis. 1997, Sep.; 25 Suppl. 2: S78-87).

As shown in Table 1, when the primers of SEQ ID NO:1 and SEQ ID NO:2were used in combination, the DNAs of bacteria of the Escherichia,Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia, Hafnia,Plesiomonas, Shigella and Yersinia genera, which are under theEnterobacteriaceae family, and of the Vibrio, Photobacterium andAeromonas genera, which are belonged to the Vibrioaceae family, weredetected.

When the primers of SEQ ID NO:3 and SEQ ID NO:4 were used incombination, the DNAs of bacteria of the Staphylococcus, Bacillus andAerococcus genera were detected.

Thus it is evident that when the primers of SEQ ID NO:1 and SEQ ID NO:2are used in combination, bacteria of the Enterobacteriaceae andVibrioaceae families are detected specifically and, when the primers ofSEQ ID NO:3 and SEQ ID NO:4 are used in combination, bacteria of theStaphylococcus, Bacillus and Aerococcus genera are specificallydetected.

Example 4 Separation of Bacteria from Foodstuffs by Centrifugation

To steamed noodles (confirmed free of Staphylococcus aureus) made fromwheat flour, starch, brine and water were added about 4.0×10⁴ cells, pergram of the noodles, of the Staphylococcus aureus IFO 3060 strain, 9times the noodle weight of phosphate buffer (pH 7.2) was added, and thenoodles were ground using a Stomachere® homogenizer.

A 1-ml portion of the ground noodle liquid A was centrifuged at 100×gfor 1 minute, the supernatant B obtained was centrifuged at 10,000×g for5 minutes, and the sediment obtained was suspended in 1 ml of brainheart infusion medium (suspension C). The bacterial counts in the groundnoodle liquid A, supernatant B and suspension C as measured were4.1×10³/ml, 4.0×10³/ml and 4.0×10³/ml, respectively. This indicatessuccessful transfer or separation of most of the bacteria initiallyadded to the noodles into the suspension C.

In addition, experiments were carried out in the same manner usingstrains of the Escherichia coli JCM 1649^(T) , Salmonella typhimuriumIFO 13245, and Bacillus cereus IFO 15305^(T). Similar results wereobtained.

Example 5 Detection Limits

<DNA Preparation>

To portions of steamed noodles (confirmed free of Staphylococcus aureus)made from wheat flour, starch, brine and water were added about 10²,10³, 10⁴ or 10⁵ cells, per gram of the noodles, of the Staphylococcusaureus IFO 3960. An amount of 9 times the noodle weight of phosphatebuffer (pH 7.2) (cf. Shokuhin no Eiseibiseibutu Kensa (Testing Foods forHealth and Microorganisms), published 1983 by Kodansha, page 440) wasadded to each portion of the noodles, and the noodles were ground usinga Stomacher® homogenizer. The bacterial counts in these ground noodleportion were about 10, 10², 10³ and 10⁴ per milliliter, respectively.

A 10-ml portion of each ground noodle liquid was centrifuged at 100×gfor 2 minute, and the supernatant was centrifuged at 3,000×g for 15minutes. The sediment was suspended in 5.0 ml of TE buffer (5 mM Tris,0.1 mM EDTA) for rinsing, the suspension was again centrifuged at3,000×g for 15 minutes, and the sediment was recovered.

DNA extraction from the sediment was effected using a DNA extraction kit(product of Gentra SYSTEMS), followed by dissolution in 20 μl of TEbuffer.

<PCR>

Sterilized water and the buffer solution for PCR were added to 2 μl ofeach DNA solution obtained in the above manner for adjustment to thefollowing final concentrations: 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl(pH 8.3), 200 μM dNTPs, 0.2 μM each primer. Thereto was added 1.25 unitsof Taq DNA polymerase (TaKaRa), and the PCR was carried out with a totalliquid amount of 50 μl.

As for the primers, the primer of SEQ ID NO:3 and the primer of SEQ IDNO:4 were used in combination.

As for the temperature conditions for PCR, thermal denaturation wascarried out at 94° C. for 30 seconds, annealing at 58° C. for 30seconds, and DNA elongation at 72° C. for 30 seconds. Under theseconditions, the PCR was carried out in 40 cycles. The PCR apparatus usedwas Geneamp PCR system 9700 (Applied Biosystems).

Each PCR product obtained was electrophoresed on 1.6% agarose gel(TaKaRa) at 100 V for 25 minutes using Mupid-2 (Cosmo Bio), the gelobtained after electrophoresis was stained with ethidium bromide (1.0μg/ml), and band observation was made under a UV lamp.

The results are shown in FIG. 3. FIG. 3 indicates that when about 10³cells per gram of noodles (about 10² cells per ml of the ground noodleliquid) of Staphylococcus aureus are present, the DNA thereof can bedetected.

As for the Escherichia coli JCM ₁₆₄₉ ^(T) , Salmonella typhimurium IFO13245 and Bacillus cereus IFO 15305^(T) as well, the DNA of each straincould be detected when about 10³ bacterial cells per gram of the noodles(about 10² cells per ml of the ground noodle liquid) were present. ForEscherichia coli and Salmonella typhimurium, the primer of SEQ ID NO:1and the primer of SEQ ID NO:2 were used in combination. For Bacilluscereus, the primer of SEQ ID NO:3 and the primer of SEQ ID NO:4 wereused in combination.

Example 6 PCR After Cell Growth

<DNA Preparation>

To portions of steamed noodles (confirmed free of Escherichia coil orBacillus cereus) made from wheat flour, starch, brine and water wereadded about 10 cells, per gram of the noodles, of the Escherichia coilJCM 1649^(T) or Bacillus cereus IFO ₁₅₃₀₅ ^(T). An amount of 9 times thenoodle weight of brain heart infusion medium was added to each portionof the bacterial cell-added noodles, and the noodles were groundaseptically using a Stomacher® homogenizer.

A 10-ml portion of each ground noodle liquid was cultured at 35° C. for5-18 hours using a shaking incubator (revolution of 200 rpm). The celldensity in the ground noodle liquid before cultivation was 1 cell/ml. Inthe case of Escherichia coli, it was 1.5×10³ cells/ml after 5 hours ofcultivation, 7.0×10³ cells/ml after 6 hours of cultivation, 5.2×10⁴cells/ml after 7 hours of cultivation, and 2.0×10⁹ cells/ml after 18hours of cultivation, as a result of bacterial cell proliferation. Inthe case of Bacillus cereus, it was 5.3×10³ cells/ml after 5 hours ofcultivation, 4.3×10⁴ cells/ml after 6 hours of cultivation, 4.1×10⁵cells/ml after 7 hours of cultivation, and 7.2×10⁸ cells/ml after 18hours of cultivation, as a result of cell proliferation.

A 1.3-ml portion of each culture fluid was collected and centrifuged at100×g for 1 minute, the supernatant was centrifuged at 10,000×g for 5minutes, and the sediment was recovered. For rinsing, this sediment wassuspended in TE buffer (5 mM Tris, 0.1 mM EDTA), the suspension wascentrifuged at 10,000×g for 5 minutes, and the sediment was recovered.

To the sediment was added 200 μl of 10% Chelex solution (10% (w/v)Chelex, 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA), the mixture was heated ona boiling water bath for 5 minutes and then centrifuged at 10,000×g for5 minutes, and the supernatant was recovered. The supernatant containedDNA, hence was used as the DNA solution for PCR.

<PCR>

Sterilized water and the buffer solution for PCR were added to 1 μl ofeach DNA solution obtained for adjustment to the following finalconcentrations: 2.0 mM MgCl₂, 50 mM Tris-HCl (pH 8.3), 0.25 mg/ml BSA,200 μM dNTPs, 0.2 μM each primer. Thereto was added 1.0 unit of HotStart Taq DNA polymerase (product of Roche Diagnostics), and the PCR wascarried out with a total liquid amount of 20 μl.

For Escherichia coli, the primers of SEQ ID NO:1 and SEQ ID NO:2 wereused in combination. For Bacillus cereus, the primers of SEQ ID NO:3 andSEQ ID NO:4 were used in combination.

As for the temperature conditions for PCR, the reaction mixture wasmaintained at 95° C. for 15 minutes and then thermal denaturation wascarried out at 95° C. for 5 seconds, annealing at 59° C. for 10 secondsand DNA elongation at 72° C. for 20 seconds. Under these conditions, thePCR was carried out in 30 cycles. The PCR apparatus used wasLightCycler® (product of Roche Diagnostics).

Each PCR product obtained was electrophoresed on 1.6% agarose gel(TaKaRa) at 100 V for 25 minutes using Mupid-2 (Cosmo Bio). The gelobtained after electrophoresis was stained with ethidium bromide (1.0μg/ml), and band observation was made under a UV lamp.

The electrophoretic patterns are shown in FIG. 4. (A) in FIG. 4 showsthe results with Escherichia coli, and (B) in FIG. 4 shows the resultswith Bacillus cereus. In the case of Escherichia coli, it is evident, asshown in FIG. 4(A), that when about 10 cells thereof are present in eachgram of the noodles, the DNA thereof can be detected by the above PCRafter 5 hours or a longer time of cultivation by the above procedure. Inthe case of Bacillus cereus, too, it is evident, as shown in FIG. 4(B),that when about 10 cells thereof are present in each gram of thenoodles, the DNA thereof can be detected by the above PCR after 5 hoursor a longer time of cultivation by the above procedure.

Further, similar results were obtained with the Salmonella typhimuriumIFO 13245 strain by the combined use of the primer of SEQ ID NO:1 andthe primer of SEQ ID NO:2, and similar results were obtained with theStaphylococcus aureus IFO 3060 strain by the combined use of the primerof SEQ ID NO:3 and the primer of SEQ ID NO:4.

INDUSTRIAL APPLICABILITY

The first primer of the invention is a primer for detecting bacteria ofthe Escherichia, Salmonella and Vibrio genera, and the second primer ofthe invention is a primer for detecting bacteria of the Staphylococcus(in particular Staphylococcus aureus) and Bacillus (in particularBacillus cereus) genera. Food-derived chloroplasts and mitochondria arenot detected by these primers. Therefore, by carrying out the PCR usingthese primers, it is possible to check the possibility of contaminationof foodstuffs by main food poisoning-causing bacteria with ease and highsensitivity.

Therefore, these primers can be suitably utilized in primary screeningin testing of foodstuffs relatively low in frequency of contaminationfor bacteria, in particular in voluntary testing for bacteria by foodmanufacturers prior to product shipment.

When, in carrying out the method of detecting bacteria according to theinvention, the food grinding step and centrifugation procedure arecarried out, bacteria are efficiently separated and collected fromfoodstuffs and, therefore, it is no longer necessary to separatebacteria from foodstuffs and cultivate the same. The testing time isthus shortened accordingly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the agarose gel electrophoretic patternsafter the PCR using the primers of the invention with type strain DNAsas templates.

FIG. 2 is a representation of the agarose gel electrophoretic patternsafter the PCR using the primers of the invention or the primers capableof detecting bacteria in general with plant-derived DNAs as templates.

FIG. 3 illustrates the bacterial detection limit in an example of thePCR using the primers of the invention.

FIG. 4 shows the necessary bacterial cultivation times in other examplesof the PCR using the primers of the invention.

1. A primer consisting of the base sequence of base numbers 1 to 30 inSEQ ID NO:1, or a primer which is a DNA comprising at most 40 bases andcontaining this base sequence on the 3′ terminal side thereof.
 2. Aprimer set, comprising: the primer of claim 1, and a primer consistingof the base sequence of bases 1 to 23 in SEQ ID NO:2, or a primer whichis a DNA comprising at most 40 bases and containing this base sequenceon the 3′ terminal side thereof.
 3. A PCR kit, comprising: the primerset of claim 2; and an additional primer set, comprising a primerconsisting of the base sequence of base numbers 1 to 24 in SEQ ID NO:3,or a primer which is a DNA comprising at most 40 bases and containingthis base sequence on the 3′ terminal side thereof and a primerconsisting of the base sequence of base numbers 1 to 33 in SEQ ID NO:4,or a primer which is a DNA comprising at most 40 bases and containingthis base sequence on the 3′ terminal side thereof.
 4. A method fordetecting bacteria which comprises the step i) of extracting DNAscontained in a foodstuff sample to be tested therefrom and the step ii)of carrying out the PCR using a primer set according to claim 2 with theDNA extracted as template and detecting the DNA thus amplified.
 5. Aprimer consisting of the base sequence of base numbers 1 to 24 in SEQ IDNO:3, or a primer which is a DNA comprising at most 40 bases andcontaining this base sequence on the 3′ terminal side thereof.
 6. Aprimer set, comprising: the primer of claim 5, and a primer consistingof the base sequence of base numbers 1 to 33 in SEQ ID NO:4, or a primerwhich is a DNA comprising at most 40 bases and containing this basesequence on the 3′ terminal side thereof.
 7. A method for detectingbacteria which comprises the step i) of extracting DNAs contained in afoodstuff sample to be tested therefrom and the step ii) of carrying outthe PCR using a primer set according to claim 6 with the DNA extractedas template and detecting the DNA thus amplified.
 8. A primer consistingof the base sequence of base numbers 1 to 33 in SEQ ID NO:4, or a primerwhich is a DNA comprising at most 40 bases and containing this basesequence on the 3′ terminal side thereof.